The epidermis at the surface of the skin provides the first line of defense for an organism. The expression of the Epidermal Differentiation Complex (EDC) genes is critical to this epidermal barrier function. The EDC encodes 4 gene families (FLG-like, LCE, SPRR, and S100) that are coordinately expressed and provide the essential cellular components that define a functional epidermal keratinocyte. It has been 20 years since the discovery of the EDC and the molecular mechanisms that govern concomitant activation of the EDC in the keratinocyte continues to elude the field. Using an unprecedented and comprehensive genomics approach to skin biology, we directly revisit this important question. Enhancers are important regulatory elements in the genome that are noncoding. Enhancers activate gene expression by proximal looping to the respective gene promoter that is mediated by transcription factor binding. Our recent studies identified a highly conserved enhancer within the EDC, namely 923, whose regulatory activity coincided with and is specific to the spatial and temporal patterning of epidermal differentiation and EDC transcription across the developing mouse epidermis. Using innovative chromosomal conformation capture methods, we further identified dynamic chromatin remodeling of the EDC with respect to the 923 enhancer during keratinocyte differentiation. A role for 923 enhancer activity for EDC activation was further supported by aberrant EDC chromatin remodeling and repressed EDC gene activation upon the loss of AP-1 transcription factor binding to 923. This led us to propose the following hypothesis. The 923 enhancer is a critical regulatory enhancer that imposes a specific configuration of the chromatin architecture for coordinate transcriptional activation of the EDC for epidermal differentiation. We propose the following aims: Aim 1. Identify the conserved epigenetic mechanisms of coordinate EDC gene expression in mice and humans. Here we will test the hypothesis that 923 is released from CTCF-bound insulators thereby allowing accessible AP-1 binding and for 923 to enhance activated EDC gene expression. We will use genomics approaches (4C-seq) to define the chromatin interactions with respect to 923, filaggrin, and a known CTCF binding site and identify the epigenetic mechanisms that govern these interactions (parallel ChIP-seq, RNA-seq, and ATAC-seq studies). Aim 2. Determine the loss of the 923 enhancer in epidermal development. We have successfully generated 5 independent lines of 923 deleted (3) and floxed mice (2) using the state-of-the-art CRISPR/Cas9-mediated genome engineering method. Molecular and cellular studies will be performed to determine the mouse phenotypes. Accomplishments of these aims will have a significant impact on our understanding of the molecular underpinnings linking chromatin architecture to gene expression at an unprecedented resolution in skin biology and relevant to all human tissue types. This study will also provide the necessary tools to develop novel strategies to correct aberrant epidermal differentiation with targeted epigenetic and chromatin remodeling therapies in medicine.